1、HCV分子生物学分子生物学陈忠斌博士陈忠斌博士军事医学科学院放射与辐射医学研究所军事医学科学院放射与辐射医学研究所电磁与激光生物学研究室电磁与激光生物学研究室The Family FlaviviridaeFields Virology,5th Edition.D.M.Knipe and P.M.Howley,Eds.Lippincott-Raven Publishers,Philadelphia(2007).Phylogenetic tree based on analysis of NS3 helicase regionsMEMBERS OF THE FAMILY FLAVIVIRIDAEG
2、enus Flavivirus Tick-borne viruses Mammalian tick-borne group(15)Tick-borne encephalitis virus,European subtype(TBEV-Eu)Tick-borne encephalitis virus,Far Eastern subtype(TBEV-FE)Seabird tick-borne group(4)Tyuleniy virus Mosquito-borne viruses Aroa virus group(4)Aroa virus Dengue virus group(5)Dengue
3、 virus,types-1 to 4(DENV-1 to DENV-4)Kedougou virus Japanese encephalitis group(10)Japanese encephalitis virus(JEV)West Nile virus(WNV)Kokobera virus group(2)Kokobera virus Ntaya virus group(6)Ntaya virus Spondweni virus group(2)Spondweni virus Yellow fever virus group(9)Yellow fever virus(YFV)Virus
4、es with no known vector Entebbe bat virus group(3)Entebbe bat virus Modoc virus group(6)Modoc virus Rio Bravo virus group(7)Rio Bravo virus Unclassified(3)Cell fusing agent virusGenus Pestivirus Bovine viral diarrhea virus 1(BVDV-1),four serotypes Bovine viral diarrhea virus 2(BVDV-2),two serotypes
5、Border disease virus,two serotypes Classical swine fever virus(CSFV),four serotypesa Pestivirus of giraffe(unclassified)Genus Hepacivirus Hepatitis C virus(HCV),six genotypes GB virus B(GBV-B)Unclassified(2)GB virus A(GBV-A),GBV-A-like viruses GB virus C(GBV-C),Hepatitis G virus(HGV)Taxonomic Unit R
6、epresentative ExamplesHepatitis C virus,HCV Flaviviridae,Hepacivirus The first hepatotropic virus was discovered by molecular cloning Biological characteristics HCV genome is a positive-sense,single strand RNA molecule,approximately 9.7Kb in length.An estimated 3%of the worlds population more than 1
7、70 million people are infected by the hepatitis C virus(HCV).Most infections become chronic:a condition that is incurable in many patients,leading to cirrhosis,end-stage liver disease and hepatocellular carcinoma.Current medical treatment options are limited,and 10,000 to 20,000 deaths a year in the
8、 United States are from hepatitis C.Indeed,chronic HCV infection is the most common cause of liver Vol 436|Issue no.7053|18 August 2005HCV是目前病毒学研究的重点之一是目前病毒学研究的重点之一Darius Moradpour,Franois Penin&Charles M.Rice.Nature Reviews Microbiology 5,453-463(June 2007)Milestone in HCV ResearchHCV 感染：感染：“现状现状”全
9、球性流行全球性流行 3%(1.7亿人口亿人口)慢性化的危险性慢性化的危险性 75%85%疾病早期肝纤维化的发生率疾病早期肝纤维化的发生率 低低 肝硬化的危险性肝硬化的危险性 在感染后在感染后20年内达年内达 10%在感染后在感染后30年内达年内达20%肝硬化相关性病死率肝硬化相关性病死率 1%5%/年年 肝硬化患者中肝细胞癌肝硬化患者中肝细胞癌 1%4%/年年的发生率的发生率1.WHO.Hepatitis C.Fact sheet no.164.2.CDC.MMWR.1998;47(RR-19):1-39.3.CDC.Hepatitis C slide kit.September 25,200
10、0.经输血和血制品传播。经输血和血制品传播。经破损的皮肤和黏膜暴露经破损的皮肤和黏膜暴露。这是目前最主要的传播方式，在某些地区，因静脉吸毒导致的HCV传播占60%至90%。使用非一次性注射器和针头、未经严格消毒的牙科器械、内镜、侵袭性操作、针刺等也是HCV传播方式之一。文身和穿皮孔也是潜在的经血传播方式。性传播性传播。与感染者性交或高危的性方式增加传播的可能性。特别是艾滋病病毒（HIV）感染者，感染HCV的危险性更高。母婴传播母婴传播。丙型肝炎病毒传播途径丙型肝炎病毒传播途径诊断、监测诊断、监测方法方法HCV血清学试验血清学试验EIA检测抗检测抗HCV、Immunoblot assayHCV-
11、RNA定性试验定性试验PCR、TMAHCV-RNA定量试验定量试验PCR、bDNAALT肝纤维化非损伤试验肝纤维化非损伤试验肝活检肝活检基因分型基因分型诊断诊断HCV感染者的最佳实验室方法感染者的最佳实验室方法National Institute of Health Consensus Development Conference Statement:Management of Hepatitis C:2002-June 10-12,200220 位患者位患者20%痊愈痊愈HCV 感染的预后感染的预后100 位位 HCV 急性感染急性感染80 位患者位患者80%持续感染持续感染24 位患者位患
12、者30%稳定，慢性，无进展稳定，慢性，无进展Adapted from Alter HF,Seeff LB.Semin Liver Dis.2000;20:17-35.28 位患者位患者28 位患者位患者 56 位患者接受抗位患者接受抗病毒治疗病毒治疗持久性应答持久性应答(50%)肝脏疾病终末期，肝细胞肝脏疾病终末期，肝细胞癌，肝脏移植，死亡癌，肝脏移植，死亡治疗失败治疗失败(50%)30%呈严重进展呈严重进展40%有不同程有不同程度的进展度的进展32 位患者位患者24 位患者位患者HCV 的特性 黄病毒科黄病毒科 有包膜有包膜,单股正链单股正链 RNA(9.6 kb)3000 氨基酸组成的多聚
13、蛋白氨基酸组成的多聚蛋白 RNA 聚合酶缺乏校正功能聚合酶缺乏校正功能 准种准种(Quasispecies)每日复制量：每日复制量：(Trillion,1012)病毒颗粒病毒颗粒HCV Genotype 1a,1b,1c 2a,2b,2c 3a,3b 4a,4b,4c,4d,4e 5a 6a 7a,7b 8a,8b 9a 10a 11aEvolutionary tree of the principal genotypes of HCV Simmonds P.J Gen Virol.2004 Nov;85(Pt 11):3173-88.Distribution of HCV sequences
14、HCV genotype in ChinaHepacivirus genome structure and expressionHCV genes,proteins,and particles Genetic organization and procession of HCV polyproteinFEBS Journal 274(2007)47054718Structures and membrane association of hepatitis C virus(HCV)proteins Darius Moradpour,Franois Penin&Charles M.Rice.Nat
15、ure Reviews Microbiology 5,453-463(June 2007)HCV NS protein structures a,NS3.The serine protease,NS4A cofactor and RNA helicase domains are shown in pink,green and blue,respectively.The serine protease and RNA helicase active site residues are indicated in red.b,NS5A domain I.Shown is a dimer,as see
16、n in the crystal structure.Individual subunits are shown in blue and green,with their C termini(that is,leading into domain II)pointing upwards.The N termini,which presumably face the membrane,are at the bottom.The purple spheres represent Zn2+ions.Disulphide bonds are indicated in red.Brackets indi
17、cate highly conserved surfaces.A basic groove,which may bind RNA,is also indicated.c,NS5B.Shown is the typical right hand model of the RdRP,with palm,fingers and thumb domains in pink,blue and green,respectively.The C-terminal region,which is not part of the RdRP,is shown in yellow.Note the extensiv
18、e interactions between the finger and thumb domains.In addition,a -hairpin is shown in purple,and active site residues Asp 220 and Asp 318 are shown in red.Brett D.Lindenbach and Charles M.Rice.Nature 436,933-938(18 August 2005)NS3(protease)NS5ANS5B(RdRp)Life cycle of hepatitis C virus(HCV)Darius Mo
19、radpour,Franois Penin&Charles M.Rice.Nature Reviews Microbiology 5,453-463(June 2007)Current model for hepatitis C virus(HCV)entry Darius Moradpour,Franois Penin&Charles M.Rice.Nature Reviews Microbiology 5,453-463(June 2007)Entry pathway of hepatitis C virus(HCV).Ploss,A.et al.Nature 457,882886(200
20、9).Nature 457,797-798(12 February 2009)occludin as a host-cell protein essential for HCV entry.as long as human CD81,SR-BI,claudin-1 and occludin were present,every cell the authors tested became infected with HCV.nature medicine volume 15|number 3|march 2009,p251Hepatitis C virus(HCV)replication co
21、mplex Darius Moradpour,Franois Penin&Charles M.Rice.Nature Reviews Microbiology 5,453-463(June 2007)In vitro and in vivo models to study HCV In vitro modelsTransient cellular expression systems Stable,transfected cell lines(constitutive or inducible expression)Replicons(subgenomic or full-length;sel
22、ectable or transient)Related viruses(for example,GBV-B(GB virus B)or BVDV(bovine viral diarrhoea virus)Retroviral pseudoparticles displaying functional HCV(hepatitis C virus)glycoproteins Recombinant infectious HCV-JFH1In vivo modelsChimpanzee(Pan troglodytes)Transgenic mice Immunodeficient mice or
23、hepatocellular reconstitution models Related viruses(for example,GBV-B or BVDV)Darius Moradpour,Franois Penin&Charles M.Rice.Nature Reviews Microbiology 5,453-463(June 2007)HCV Cell culture No efficient,robust HCV cell culture.Infection of primary human liver cells and other cell lines with HCV-posi
24、tive serum is possible,but with low efficiency（0.01-0.1 RNA genome/cell）.HCV Replicon SystemFEBS Journal 274(2007)47054718Weakness of HCV Replicon System No all HCV genotype can be used to make efficient replicon system.1b (Lohmann V,et al.Science 1999,285:110-113)1a (Blight KJ,et al.JV 2003,77:3181
25、-3190;Y M,et al.JV 2004,78:7904-7915;)Replicon only replicates efficiently with adaptive mutations Do not produce infection virions.Not suitable for analysis of complete viral life cycle.Production of HCV pseudoparticlesFEBS Journal 274(2007)47054718The HCVcc modelFEBS Journal 274(2007)47054718FEBS
26、Journal 274(2007)47054718 Robust HCV cell culture system with HCV JFH-1 RNA and Huh7 cell.Breakthrough of HCV Research:HCV Cell Culture SystemHCV JFH-1（Japan fulminant hepatitis）Isolated from a Japanese patient with fulminant hepatitis.Genotype:2a HCV JFH1 replicon replicates to very high levels in
27、Huh7 cells without cell culture adaptive mutation.Hepatocyte:Huh7,HepG2;IMY-N9;Nonhepatic cells:Hela,HEK 293Kato,T,et al.Gastroenterology,2003,125:1808-1817Kato,T,et al.J Virol.2005,79:592-596HCV infection kinetics and passage in tissue cellsD5 from AKinetics of JFH1 HCV infection in Huh7.5.1 and Hu
28、h7Standard Therapy for HCV:Interferon-and ribavirin a 48-week(1 year)course of therapyAnti-HCV Drugs:R&DNature Reviews Drug Discovery,Vol 5,Dec.2007:991Different interferon(IFN)signalling pathways activatedby dsRNA and virusesReceptor activation or ligandreceptor complex assembled by type I,type II
29、or type II interferonsDisadvantage of Standard Therapy for HCVStandard therapy for HCV,a 48-week course of interferon-and ribavirin,can cure about half of patients,but its a very hard treatment.Along with extreme fatigue and flu-like symptoms,patients often suffer persistent headaches,muscle aches a
30、nd anemia.“Some of our patients whove gone through cancer chemotherapy say that cancer chemotherapy is easier than hepatitis C treatment,”.Interferon treatment can lead to depression and,in rare instances,suicide.Because of a combination of nondiagnosis,treatment failure and treatment avoidance,and
31、discontinuation,“only approximately 2%of the people with HCV in America are successfully treated with standard of care,”.Limitations of current treatment options:low sustained virological response(SVR)-Genotype,Certain patient populations,patients co-infected with HIV.New therapies are clearly neede
32、d.Evolution of HCV therapyNature Reviews Drug Discovery 7,799-800(October 2008)Nature Reviews Drug Discovery,Vol 5,Dec.2007:991NS5B RNA replicase inhibitors Nature Reviews Drug Discovery 6,1001-1018(December 2007)NS5B RNA replicase inhibitors:part 2.Nature Reviews Drug Discovery 6,1001-1018(December
33、 2007)NS3 hepatitis C virus(HCV)protease inhibitors Nature Reviews Drug Discovery 6,1001-1018(December 2007)Nature Reviews Drug Discovery 9,501-503(July 2010)|Nature Reviews Drug Discovery,Vol 5,Dec.2007:9912007 was a good year for hepatitis Cfor the virus,that is.Companies,including seven biotechs,
34、abandoned or suspended at least eight antiviral drugs in clinical development over the course of the year.Despite the recent drug trial failures,industry activity is still on the rise.Nature Medicine,Dec 2011,17(12)Nature Medicine,Dec 2011,17(12)New HCV drugs trigger race for more tolerable therapie
35、sImportant research and commercial milestones in telaprevir developmentNature Biotechnology volume 29 number 11 november 2011 HCV Vaccine Michael Houghton and Sergio Abrignani.Nature 436,961-966(18 August 2005)Prophylactic HCV vaccine candidates HCV immunotherapeutic vaccine candidates Michael Houghton and Sergio Abrignani.Nature 436,961-966(18 August 2005)复习题 简述HCV病毒基因组和蛋白质组特征 简述HCV复制周期以及可能的抗病毒药物靶点 简要分析HCV药物和疫苗研究的现状和前景
